With the long term objective of understanding ubiquitin-dependent protein homeostasis in the brain, the current proposal focuses on CHIP, an important ubiquitin ligase recently shown to suppress neurodegeneration caused by a variety of misfolded disease proteins. Aim 1 explores the mechanisms regulating ubiquitin chain formation by CHIP. In vitro ubiquitination assays will systematically test a range of candidate CHIP interactors for their ability to regulate the types of ubiquitin chain linkages formed by CHIP;tested candidates will include all known ubiquitin conjugating enzymes (E2s) as well as a panel of selected de ubiquitinating enzymes (DUBs) and Ubiquitin Interacting Motif-containing proteins (UIMs). These studies will determine which E2s function with CHIP, what types of linkages of ubiquitin chains are made by each E2-CHIP pair, and what role DUBs and UIM-containing proteins may play in restricting CHIP to the formation of specific types of ubiquitin chain linkages. Aim 2 is complementary to Aim 1 in that it seeks to identify proteins that interact, directly or indirectly, with CHIP in maintaining proper protein homeostasis in the brain. Two parallel approaches will be employed: 1) yeast two-hybrid analysis coupled with confirmatory co-immunoprecipitation assays;and 2 )chromatographic separation and isolation of CHIP protein complexes from wild type mice and from a polyglutamine neurodegenerative disease mouse model. Identifying the components of functional CHIP complexes in normal and neurodegenerative disease brain is a first step towards understanding the diverse roles CHIP may play in providing neuroprotection. Together, the studies in aims 1 and 2 will comprise the first comprehensive analysis of what constitutes a fully functional CHIP ubiquitin ligase. Aim 3 will establish and perform a high-throughput screen to identify compounds that stimulate CHIP activity, as enhancers of CHIP activity are predicted to boost its neuroprotective action. This will be accomplished by establishing a biochemical FRET based quenching assay to do high- throughput screening for compounds that decrease fluorescein emission. Compounds that stimulate CHIP activity may be developed into a new class of therapeutics. This proposal will provide insight into the mechanism by which CHIP maintains neuronal protein homeostasis in various neurodegenerative diseases. CHIP, an important ubiquitin ligase in the brain, has been implicated as a key regulator of brain degeneration in various animal models of human disease. The current project seeks both to understand how CHIP mediates neuroprotection and to identify compounds that enhance this activity, as they could represent new therapeutic agents for various neurodegenerative diseases.